Oct 09, 2023

Public workspaceColony PCR

DOI
dx.doi.org/10.17504/protocols.io.5jyl8pex8g2w/v1
  • 1National University of Singapore
NUS iGEM
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl8pex8g2w/v1
Protocol Citation: NUS iGEM 2023. Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pex8g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 28, 2023
Last Modified: October 09, 2023
Protocol Integer ID: 88552
Keywords: Colony PCR, PCR, Polymerase Chain Reaction, Sequencing, Gibson Assembly
Abstract
2023 NUS-Singapore iGEM team followed this protocol to perform preliminary screening of plasmid-containing colonies without the need for overnight cell culture. This screening aimed to confirm the correct assembly of DNA fragments during Gibson Assembly or the presence of target DNA fragments in the plasmid. After obtaining the results of the colony PCR, the team could then decide whether specific colonies required overnight culturing. This would allow them to perform plasmid extraction the next day, obtain the fully assembled plasmid, and send it out for sequencing to confirm the final structure.
Materials
KOD OneTM PCR Master Mix (Blue)
Safety warnings
Attention
  • Proper lab PPE must be worn at all times.
  • Since cells are used in this protocol, a Biosafety Cabinet (BSC) is required to ensure safety.
Circle the colony of interest.
Prepare the mixture below in a PCR tube, the final volume is Amount50 µL :

ItemVolume
KOD OneTM PCR Master Mix (Blue)25uL
Forward Primer1.5uL
Reverse Primer1.5uL
DI Water22uL

Mix the solution well by vortexing the tubes and then split the solution equally into 5 new PCR tubes (each PCR tube would contain Amount10 µL of the solution).

Use an inoculation loop to inoculate some cells from the colony of interest and dunk the inoculation loop into the PCR tube according to the labels.
Run a PCR with the setting below:

TemperatureDuration
98°C10s
55°C5s
68°C1 minute
Go to step 1, repeat the cycle 25 times

After the PCR is complete, proceed to run gel electrophoresis for the samples.
After gel electrophoresis is finished, put the agarose gel onto a UV Sample Tray, next, put it into the Gel Doc EZ System, and then run the gel imaging program on the desktop.
Check if the expected band occurs.
Save the gel image and discard the gel into the biohazard bin.